Purkinje fibers play a key role in normal activation of the heart, and they may be equally important in generating some cardiac arrhythmias. The long term goal of this research is a thorough understanding of excitation and excitation-contraction coupling in cardiac Purkinje fibers, particularly those mechanisms involved in arrhythmogenesis. The specific aims of this application are: 1) to analyze the slow inward current (Isi) of Purkinje fibers; 2) to determine the relations between Isi and contraction; 3) to find out whether observed relations between tension and the transient inward current (TI) and between tension and a transient outward current (Iqr) are best explained by calcium regulated membrane channels or other mechanisms; and 4) to analyze outward membrane currents in a preparation which has wide intercellular clefts, and thus few complications due to accumulation or depletion of ions in the clefts between cells. We plan to use sheep Purkinje fibers for aims 1-3, and rabbit Purkinje fibers for aim 4. The two microelectode voltage clamp technique will be used to examine the relations between membrane voltage, membrane ionic current, and isometric contraction. Isi will be separated from other currents by a combination of pharmacological, ion substitution, and kinetic analysis techniques, with due regard for the limitations of such methods and of cardiac voltage clamp techniques. This work is a continuation of a determined step by step approach to this problem we started several years ago. Experiments to accomplish the other aims are described in detail. In addition to this work, we plan a feasibility study with Dr. J. Patlak, to see if a modified patch electrode technique can be applied to Purkinje cells. With this method, the membrane patch is 'excised' from the cell, allowing a voltage clamp with perfusion of the cytoplasmic membrane face and direct observation of membrane channel properties.